Some chemical factors regulating the growth of <Emphasis Type="Italic">Lyngbya majuscula</Emphasis> in Moreton Bay,Australia: importance of sewage discharges

The relative importance of the additions of iron (Fe), phosphorus (P) and the chelating agent EDTA in promoting the growth of Lyngbya majuscula in waters collected from the NW region of Moreton Bay was investigated using a series of continuous-flow growth studies. In addition, the possible impact of sewage/waste-water treatment plant (WWTP) discharges on the growth of L. majuscula was investigated in a series of batch and semi-continuous-flow growth studies. A preliminary study on the potential of phytoplankton growth in the receiving waters to affect the productivity of L. majuscula was also conducted. The results from the continuous-flow growth studies show that the growth rates of L. majuscula in the NW Moreton Bay waters were stimulated by the addition of EDTA alone but were not stimulated by the addition of P. The additions of P + EDTA, FeEDTA and P + FeEDTA did not result in higher growth rates than those obtained by the addition of EDTA alone. These results demonstrate that the productivity of L. majuscula in the NW Moreton Bay waters was not limited by P and that the addition of Fe did not affect the productivity. The stimulation effect of EDTA could be due to various reasons but we hypothesise that the principal reason for the stimulation is that EDTA increases the bioavailability of non-labile Fe species already present in the water. The results of the batch and semi-continuous-flow growth studies show that diluted (100:1) WWTP discharge water and the receiving waters impacted by WWTP discharges supported significant growth of L. majuscula and that the addition of EDTA to those waters increased the growth potential of L. majuscula. Also the growth of phytoplankton in a sample of the receiving waters impacted by WWTP discharges significantly reduced the growth rate of L. majuscula but the addition of EDTA restored the growth rate to near its maximum value. These results suggest that phytoplankton growth (and probably that of the associated bacterioplankton) could reduce the bioavailability of trace chemical factors needed for the growth of L. majuscula in Moreton Bay and in particular, could reduce the bioavailability of Fe. Overall the results support the hypothesis that growth of L. majuscula in Moreton Bay is often limited by the bioavailability of Fe and the principal reason for this is the lack of supply of suitable organic ligands/chelators, not the lack of Fe per se. Handling editor: D. Hamilton     

Isolation and biochemical characterization of cell wall tight protein complex involved in self-flocculation of Kluyveromyces bulgaricus

Flocculation of yeasts is a cell–cell aggregation phenomenon which is driven by interactions between cell wall lectins and cell wall heteropolysaccharides. In Sabouraud medium, Kluyveromyces bulgaricus was highly flocculent. Incubation of flocculent K. bulgaricus cells with EDTA or Hecameg® led to extracts showing hemagglutinating and flocculating properties. Purification of the extracts by native PAGE gave two bands which allowed flocculation of deflocculated K. bulgaricus. Both bands with specific reflocculating activity were composed of five subunits, of which only three possessed weak reflocculating activity upon deflocculated yeast. The mixture of these three proteins allow the recovery of initial specific reflocculating activity of the complex. These three proteins, denoted p28, p36 and p48, presented, in their first 15 amino acids, homologies with glycolysis enzymes, i.e., 3-phosphoglycerate mutase, glyceraldehyde-3-phosphate dehydrogenase and enolase, respectively. However, no such enzymatic activity could be detected in the crude extract issued from treatment with EDTA and Hecameg® of flocculent yeast cells. When yeasts had grown in glucose poor medium, flocculation was drastically affected. The EDTA and Hecameg® crude extracts showed weak reflocculating activity. After PAGE, the protein complexes did not appear in the EDTA extract, but they did appear in the Hecameg® crude extract. These results suggest that: (i) self-flocculation of K. bulgaricus depends on the expression of different floc-forming protein complex, (ii) these proteins are galactose specific lectins showing homologies in their primary structure with glycolysis enzymes.     

Comparative studies on the mycorrhization of Larix decidua and Picea abies by Suillus grevillei

Summary Mycorrhization of Picea abies has been achieved, for the first time, with six strains of Suillus grevillei by a new culture method, using activated charcoal paper and liquid medium as a substrate. Mycorrhization of P. abies and Larix decidua was compared, and the process was found to be significantly different in the two tree species. S. grevillei is not incompatible with P. abies, but it forms mycorrhizae more readily with L. decidua. Hyphal growth was clearly stimulated on the surface of roots of Larix but retarded on Picea. A well organized Hartig net was formed with both tree species, but wall protuberances were frequently observed on the outer cell walls of Picea cortex cells when the Hartig net was not fully developed. No conspicuous cell wall reactions occurred in Larix roots. Cell wall protuberances may be comparable to those in transfer cells and are interpreted as an alternative to Hartig net development. Anatomical differences between roots of Larix and Picea, and physiologically active substances such as recognition factors on the root surfaces, are discussed with respect to their responsibility for the different reactions of S. grevillei.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

Enhancement of nitric oxide solubility using Fe(II)EDTA and its removal by green algae <Emphasis Type="Italic">Scenedesmus</Emphasis> sp.

A photoautotrophic cultivation of green algae Scenedesmus cells was used for the removal of nitric oxide (NO) from a model flue gas mixture. In an attempt to improve the solubility of NO in the culture broth, the addition of Fe(II)EDTA to the cultivation was investigated. The addition of Fe(II)EDTA greatly enhanced NO-dissolution in the culture broth and subsequently increased the algal-uptake of NO. NO was assimilated as a source of nitrogen for the growth of Scenedesmus cells since there was a steady increase in cell density with no other nitrogen source in the culture except the incoming NO. 40–45% of NO removal was maintained for more than 12 days with the addition of 5 mM Fe(II)EDTA in a 1-L air-lift type photobioreactor system fed with 300 ppm of NO gas at a rate of 0.3 wm. However, the NO-dissolution-enhancing capacity of Fe(II)EDTA did not reach its full potential due to its oxidation to Fe(III)EDTA, possibly induced by molecular oxygen that evolved from algal photosynthesis, and subsequent loss of chelating capabilities.     

Immunological evidence for the involvement of cell wall proteins in phosphate uptake in the yeast Saccharomyces cerevisiae

Immunological cross-reactivity between cell wall proteins obtained from two yeast genera (Candida tropicalis and Saccharomyces cerevisiae) is reported. Specific retention of two cell wall proteins from Saccharomyces cerevisiae by an immunoabsorbent column coupled with antibodies against phosphate binding protein 2 (PiBP2) from Candida tropicalis allowed to generate antibodies against the proteins from S. cerevisiae. These antibodies were effective in inhibiting phosphate uptake by S. cerevisiae cells. The proteins from S. cerevisiae displayed a phosphate binding activity which was inhibited in the presence of the forementioned antibodies. These results and the observation that the amount of these proteins in the shock fluid was dependent of the growth conditions (i.e., in the presence or in the absence of phosphate) support the idea that these proteins are involved in the high affinity phosphate transport system.Abbreviations Pi inorganic phosphate - PiBP2 phosphate binding protein 2 obtained from Candida tropicalis - Tris Tris(hydroxymethyl)-aminoethane - MES [2-(N-Morpholino)] ethanesulfonic acid - EDTA ethylene diamine tetraacetic acid, disoldium salt - PMSF phenylmethyl sulfonyl fluoride - SDS sodium dodecyl sulfate - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Inhibition on <Emphasis Type="Italic">Candida albicans</Emphasis> biofilm formation using divalent cation chelators (EDTA)

Candida albicans can readily form biofilms on both inanimate and biological surfaces. In this study we investigated a means of inhibiting biofilm formation using EDTA (Ethylenediaminetetra-acetic acid), a divalent cation chelating agent, which has been shown to affect C. albicans filamentation. Candida albicans biofilms were formed in 96-well microtitre plates. Cells were allowed to adhere for 1, 2, and 4 h at 37°C, washed in PBS, and then treated with different concentrations of EDTA (0, 2.5, 25, and 250 mM). EDTA was also added to the standardized suspension prior to adding to the microtiter plate and to a preformed 24 h biofilm. All plates were then incubated at 37°C for an additional 24 h to allow for biofilm formation. The extent and characteristics of biofilm formation were then microscopically assessed and with a semi-quantitative colorimetric technique based on the use of an XTT-reduction assay. Northern blot analysis of the hyphal wall protein (HWP1) expression was also monitored in planktonic and biofilm cells treated with EDTA. Microscopic analysis and colorimetric readings revealed that filamentation and biofilm formation were inhibited by EDTA in a concentration dependant manner. However, preformed biofilms were minimally affected by EDTA (maximum of 31% reduction at 250 mM). The HWP1 gene expression was reduced in EDTA-treated planktonic and biofilm samples. These results indicate that EDTA inhibits C. albicans biofilm formation are most likely through its inhibitory effect on filamentation and indicates the potential therapeutic effects of EDTA. This compound may serve a non-toxic means of preventing biofilm formation on infections with a C. albicans biofilm etiology.     

Characteristics of copper tolerance in Yarrowia lipolytica

We discovered that a mutant strain of the dimorphic yeast Yarrowia lipolytica could grow in the yeast form in high concentrations of copper sulfate. The amount of metal accumulated by Y. lipolytica increased with increasing copper concentrations in the medium. Washing with 100 mM EDTA released at least 60% of the total metal from the cells, but about 20–25 μmol/g DW persisted, which represented about 30% of the soluble fraction of cultured cells. The soluble fraction (mainly cytosol) contained only about 10% of the total metal content within cells cultured in medium supplemented with 6 mM copper. We suggest that although a high copper concentration induces an efflux mechanism, the released copper becomes entrapped in the periplasm and in other parts of the cell wall. Washing with EDTA liberated not only copper ions, but also melanin, a brown pigment that can bind metal and which located at the cell wall. These findings indicated that melanin participates in the mechanism of metal accumulation. Culture in medium supplemented with copper obviously enhanced the activities of Cu, Zn-SOD, but not of Mn-SOD.     

Ultrastructural and Chemical Alteration of the Cell Envelope of Pseudomonas aeruginosa, Associated with Resistance to Ethylenediaminetetraacetate Resulting from Growth in a Mg2+-Deficient Medium

Cells of Pseudomonas aeruginosa became resistant to the lytic effect of ethylenediametetraacetate (EDTA) when grown in a Mg(2+)-deficient medium. To correlate ultrastructural changes in the cell wall associated with the shift to EDTA-resistance, a freeze-etch study was performed. Upon fracturing, the outer cell wall membrane split down the hydrophobic center to reveal the outer (concave) and inner (convex) layers. The concave cell wall layer of EDTA-sensitive cells grown in Mg(2+)-sufficient medium contained spherical units resting on an underlying smooth support layer. Upon EDTA treatment, approximately one-half of these spherical units were extracted. Cells grown in Mg(2+)-deficient medium were resistant to EDTA. The concave cell wall layer of EDTA-resistant cells had increased numbers of highly compacted spherical units, giving this layer a disorganized appearance. The highly compacted appearance of this layer was unaltered by EDTA treatment. Thus, growth in Mg(2+)-deficient medium resulted in cells which were resistant to EDTA and which possessed an ultrastructurally altered outer layer of the outer cell wall membrane. Cell envelopes from EDTA-resistant cells were found to possess 18% less phosphorus, 16.4% more total carbohydrate, and 13.3% more 2-keto-3-deoxyoctonate than cell envelopes from EDTA-sensitive cells. There were also qualitative, but not quantitative, differences in the protein content of cell envelopes from EDTA-resistant and EDTA-sensitive cells.     

Monoclonal antibodies as molecular probes for cell wall antigens of the brown alga,Fucus

Monoclonal antibodies to cell wall carbohydrates were produced against carbohydrates extracted from the brown alga, Fucus distichus ssp. edentatus (de la Pyl.) Powell. Mouse spleen cells were immunized in vitro with alginate and fucans, and hybridoma cultures were screened by enzyme immunoassay. Most antibodies were immunoglobulin (Ig)M and one was IgA. Antigens were localized on methacrylate sections of Fucus tissues by indirect immunofluorescence. Each antibody labelled tissues with a distinctive distribution pattern in cell walls and extracellular matrix regions, demonstrating that each antibody was specific for a different extracellular epitope (i.e., antigenic determinant). Most antibodies also labelled intracellularly on at least one cell type. Punctate, fibrous or clumped labelling was characteristic of individual antibodies and provided information related to carbohydrate structure and solubility. These antibodies are molecular probes for small regions on cell wall polymers and should be valuable in studies of cell wall synthesis, secretion, assembly and modification as well as carbohydrate fine structure and function.Abbreviations EDTA ethylenediaminetetraacetic acid - EIA enzyme immunoassay - Ig immunoglobulin (IgG, IgM and IgA are immunoglobulin types)     

Synthesis and turnover of cell body-agglutinin as a pool of flagellar surface-agglutinin inChlamydomonas reinhardii gamete

Chlamydomonas reinhardii cells were broken in a French press and the soluble fraction was tested for agglutination activity. Deflagellated cell bodies ofmt ⁺ andmt ^- gametes yielded soluble fractions that were able to isoagglutinate gametes of the opposite mating type. When the wild-type gametes of opposite mating types were mixed, the cell body-agglutinins were used up during flagellar agglutination and subsequent cell fusion. When thefus mt ⁺ andmt ^- gametes agglutinated without successive fusion, the amount of cell body-agglutinins sharply decreased, then increased and reached the premixing level: the recovery was blocked by cycloheximide. When cells were treated with EDTA or trypsin, the cell body-agglutinins as well as flagellar surface-agglutinins were completely lost without apparent loss of motility. The EDTA extract contained the same amount of agglutinins as observed in the cell bodies before extraction, and this amount was about 100 times higher than that in the EDTA extract of isolated flagella. By the addition of trypsin inhibitor, the trypsinized gametes resynthesized the cell body-agglutinins. The process was sensitive to cycloheximide in both mating type gametes and to tunicamycin inmt ⁺ gametes.Abbreviations mt ^+/- mating type plus or minus - CHI cycloheximide - TI trypsin inhibitor - TM tunicamycin     

Export of porin to the outer membrane of the phototrophic bacterium Rhodobacter capsulatus 37B4

Export of porin to the outer membrane of the phototrophic purple bacterium Rhodobacter capsulatus was studied with the use of the uncoupler of the electron transport chain, carbonylcyanide-m-chlorophenylhydrazone (CCCP). The agent reversibly blocked the transport of porin across the cytoplasmic membrane. By means of radioactive labeling and immunoprecipitation, porin was found to occur in two forms: (i) the exported form that was extractable from the outer membrane without disrupting the cells, and (ii) a pre-form with a slightly higher apparent molecular mass which accumulated in the cells during the block of the export process. Proteolysis studies revealed that the preform was highly sensitive to added proteases, whereas the exported form was resistant.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - DMSO dimethylsulfoxide - EDTA ethylenediamine tetraacetic acid - OMP outer membrane porin; pre-OMP, form of outer membrane porin before export - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride - SDS sodium dodecyl sulfate     

Cytoplasmic reorganization accompanies the deposition of a bipolar cell wall in the large-celled red alga Anotrichium tenue

The filamentous red alga Anotrichium tenue C. Aghard (Naegeli) (formerly Griffithsia tenuis C. Aghard; Baldock, 1976, Aust. T. Bot. 24, 509–593) has large (1–2 mm long), cylindrical, multinucleate cells that exhibit a daily, cyclic redistribution of chloroplasts. Chloroplasts accumulate in the mid-region of each growing cell during the day; consequently, filaments appear banded with a light apical end-band, a dark mid-band and a light basal end-band in each growing cell. Chloroplasts disperse at night so that the bands are no longer visible and the cells appear evenly pigmented. Anotrichium tenue also has a type of cell elongation, known as bipolar band growth, in which new material is added to the microfibrillar part of the wall in bands located at the apical and basal poles of elongating cells. This site of wall growth corresponds to the position of the light-colored end-bands present during the day. Here we examine the structural relationship between the cytoplasmic bands and the wall-growth bands. Our results show that, in addition to the previously described bipolar wall bands, there is a non-microfibrillar wall band in the mid-region of the cell. This wall component apparently branches from near the top of the microfibrillar outer wall and terminates near but not at the bottom of the cell. It contains nodules of sulphated polysaccharide material secreted from a band of vesicles, which co-localize with the chloroplasts in the mid-band. The outer wall appears to enclose the entire cell. Nuclei do not redistribute with the chloroplasts or wall vesicles into the mid-band but remain evenly distributed throughout the cytoplasm. Each wall component grows by a different mechanism. We show that two types of wall growth, diffuse and the bipolar-type of tip growth, occur in the same cell and we propose that the observed segregation of the cytoplasm supports localized growth of the unique inner wall component. Additionally, we show that A. tenue is an excellent model for study of the role and mechanism of cytoplasmic compartmentalization and cell polarity during plant cell growth.We wish to thank Dr. Richard Cloney (University of Washington and Dr. Tom Schroeder (Friday Harbor Laboratories, Friday Harbor, Wash.) for helpful discussions and critical review of this work. We also thank Dr. Susan Waaland (University of Washington) for sharing her original observations on the chloroplast banding phenomenon in Anotrichium tenue. We are grateful to the Friday Harbor Laboratories for the use of their space and facilities. This research was supported by funds from the Washington Sea Grant Program (awarded to J.R.W.) and by the Developmental Biology Training Grant, predoctoral fellowship, National Institutes of Health, No. HD07183 to A.W.S.     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Differences in the virulence of Heterorhabditis bacteriophora and Steinernema scarabaei to three white grub species: The relative contribution of the nematodes and their symbiotic bacteria

Because susceptibility of white grub species to entomopathogenic nematodes differs, we compared the virulence of Photorhabdus temperata and Xenorhabdus koppenhoeferi, the symbiotic bacteria of the nematodes Heterorhabditis bacteriophora and Steinernema scarabaei, respectively, to the three white grub species, Popillia japonica, Rhizotrogus majalis, and Cyclocephala borealis. Both bacteria were pathogenic to all three grub species even at 2 cells/grub. However, the median lethal dose at 48 h post injection and median lethal time at 20 cells/grub showed that P. temperata was more virulent than X. koppenhoeferi to C. borealis. Although H. bacteriophora is less pathogenic than S. scarabaei to R. majalis and P. japonica, their symbiotic bacteria did not differ in virulence against these two grub species, and they also showed similar growth patterns both in vitro and inside R. majalis larvae at 20 °C. We then tested the pathogenicity of oral- and intrahemocoel-introduced H. bacteriophora to R. majalis to determine whether nematodes are able to successfully vector the bacteria into the hemolymph. Hemocoel injected H. bacteriophora was pathogenic to R. majalis indicating successful bacterial release, but orally introduced H. bacteriophora were not. Dissection of grubs confirmed that the orally introduced H. bacteriophora were unable to penetrate into the hemolymph through the gut wall. We conclude that the low susceptibility of R. majalis to H. bacteriophora is not due to the symbiotic bacteria but rather to the nematode’s poor ability to penetrate through the gut wall and the cuticle to vector the bacteria into the hemolymph.     

Survival of Chromatium vinosum at low light intensities

The effect of low irradiation on the viability of Chromatium vinosum was investigated. Cultures were precultivated at 1,000 lux (=0.1/h). Then, before the substrate was depleted, illumination was changed to either complete darkness or about 30 lux. Previously, the latter light intensity had been found not to promote growth.The parameters assayed were viability, protein, bacteriochlorophyll, ATP, RNA, DNA, absorbance (E ₂₆₀) of the supernatant, and total anthron-positive material.The data show that irradiation insufficiently high to promote growth, results in viability percentages as high as 90% after 8 days, whereas cultures incubated in complete darkness are virtually dead by then. Neither in the light nor in the dark a degradation of protein or cell wall hexoses was observed. The RNA content also remained constant. However, particularly in the dark cultures DNA was found to decrease concomitant with increased E ₂₆₀ readings of the supernatant. It is considered unlikely that such essential macromolecules are degraded to serve the maintenance energy requirements. The ecological impact of the observations is discussed.Non-Standard Abbreviations PHB poly--hydroxybutyric acid - Bchl Bacteriochlorophyll     

Two water molds can grow without measurable turgor pressure

The water molds Achlya bisexualis Coker and Saprolegnia ferax (Gruithuisen) Thuret (Class: Oomycetes) normally grow in the form of slender hyphae with up to 0.8 MPa (8 bar) of internal pressure. Models of plant cell growth indicate that this turgor pressure drives the expansion of the cell wall. However, under conditions of prolonged osmotic stress, these species were able to grow in the absence of measurable turgor. Unpressurized cells of A. bisexualis grew in the form of a plasmodium-like colony on solid media, and produced a multinucleate yeast-like phase in liquid. By contrast, the morphology of S. ferax was unaffected by the loss of turgor, and the mold continued to generate tip-growing hyphae. Measurements of cell wall strength indicate that these microorganisms produce a very fluid wall in the region of surface growth, circumventing the usual requirement for turgor.Abbreviations DAPI 4,6-diamidino-2-phenylindole - PEG polyethylene glycol This work was supported by National Science Foundation grant DCB 90-17130.     

Cellular expression patterns of acetylcholinesterase activity during grasshopper development

We examined the expression of acetylcholinesterase (AChE) in the nervous system and epidermal body structures during embryonic and larval development of two grasshopper species: Locusta migratoria and Schistocerca americana. Histochemical labelling was blocked by the enzyme inhibitors eserine and BW284c51, but not by iso-OMPA, showing that the staining reflected true AChE activity. The majority of staining was localized on the cell surface but granular intracellular staining was also visible in many cell bodies. In both species, the cellular expression of AChE followed a similar but complex spatiotemporal staining pattern. Initially, mainly epidermal tissue structures were stained in the various body appendages (stages 25%–30%). Labelling subsequently appeared in outgrowing neurons of the central nervous system (CNS) and in the nerves innervating the limbs and dorsal body wall (stages 30%–40%). The latter staining originated in motoneurons of the ventral nerve cord. In a third phase (after 45%), the somata of certain identified mechanosensory neurons started to express AChE activity, presumably reflecting cholinergic differentiation. Staining was also found in repo-positive glial cells of the CNS, longitudinal glia of connectives, glia of the stomatogastric nervous system and glial cells ensheathing peripheral nerves. Glial cells remained AChE-positive during larval to adult development, whereas motoneurons lost their AChE expression. The expression pattern in non-neuronal cells and glutamatergic motoneurons and the developmental appearance of AChE prior to synaptogenesis in the CNS suggest non-cholinergic functions of AChE during grasshopper embryogenesis. Financial support was provided by the Deutsche Forschungsgemeinschaft (Bi 262/7-1 and 262/11-1)     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

The apparent oxidation of NADH by whole cells of the methylotrophic bacterium Methylophilus methylotrophus

Previous reports that whole cells of Methylophilus methylotrophus oxidase exogenous NADH have been investigated. Essentially identical rates of oxygen consumption were observed following the addition of methanol or NADH to whole cells. Both activities were inhibited by EDTA and hydroxylamine, but not by HQNO, and exhibited similar pH optima. Analyses of the reaction stoichiometry with NADH as substrate showed that the expected amount of oxygen was consumed, but also revealed acidification (instead of alkalinisation) and no oxidation of NADH. Further studies showed that commerical NADH is contaminated with ethanol which is oxidised to acetic acid by the low specificity methanol oxidase system present in this organism. The oxidation of exogenous NADH by whole cells of M. methylotrophus reported previously is therefore spurious.Abbreviations EDTA = ethylenediaminetetracetate - HQNO = 2–n-heptyl-4-hydroxy-quinoline-N-oxide - DEAE = diethylaminoethyl